One of the most recent applications of synthetic oligonucleotides is their use in microarray technology. DNA microarray is a powerful tool, which allows the simultaneous detection of many different target molecules present in a sample. The technology deals with the covalent fixing of oligonucleotides (small fragments of DNA), cDNA, PCR fragments, etc on the solid or polymer surfaces. These DNA chips or bio-chips can be used for mutation detection, SNP analysis, disease diagnosis, monitoring of gene expression, etc. DNA Microarrays have extended the basic technique by using much smaller amounts of DNA probe, and more importantly by allowing researchers to perform tens of thousands of hybridization experiments in parallel. This allows researchers to view the response of whole genomes to various stimuli.
A number of methods have been reported for the preparation of oligonucleotide arrays. In general, there are some pre-requisites for the preparation and applications of oligonucleotide arrays: (a) arrays require the parallel preparation of a large number of oligonucleotides or the immobilization of a large number of previously prepared oligonucleotides, (b) each immobilized sequence must be addressable, (c) each immobilized oligomer sequence must be accessible to interaction with target biomolecule, e.g. to hybridization with analyte nucleic acids, and (d) this interaction must be susceptible to monitoring.
Basically, oligonucleotide arrays are solid surfaces consisting of hundreds or thousands of oligomers covalently attached at discrete locations, which are available for hybridization. Currently, two methods are being followed for the construction of oligonucleotide arrays. The first one involves direct synthesis of oligonucleotides at the pre-determined sites on the solid or polymeric surfaces using photolithographic technique (Fodor et al., Science (251 (1991) 767). The second method is based on the immobilization of pre-synthesized oligonucleotides on the solid or polymeric surfaces using a suitable hetero- or homobifunctional reagent or a coupling reagent (Beier and Hoheisel, Nucl. Acids Res., 27 (1999) 1970). Alternatively, this can be achieved also by direct reaction between two functionally active groups present on the surface and oligomer moiety. The later method provides flexibility in the sense that it does not require expensive chemistry and sophisticated instrumentation. Modifications can easily be incorporated in the oligomers according to the functionalities present on the solid surface. This method is preferred for generating low to moderate density oligonucleotide arrays.
Generally, in the preparation of oligonucleotide arrays by post-synthesis immobilization, the following chemical steps are needed to be discussed: (a) the choice of substrate material and its primary functionalization, (b) the synthesis of oligonucleotides with specific functional groups, (c) the activation of the substrate functionality, (d) the activation of the oligonucleotide terminal group, and (e) the immobilization reaction, i.e. the reaction of the activated substrate and oligonucleotide.
Several surface materials have been tested so far, such as nylon, nitrocellulose, polypropylene, polystyrene, silicon, glass, teflon, etc. Out of these, glass and polypropylene stand a good chance because these materials can easily be derivatized to generate functional groups on the surface, viz., aminoalkyl, carboxyl, aldehyde, mercaptoalkyl, etc. Glass has an additional advantage in that the currently used laser scanners can also be used.
Most of the immobilization reactions involve the attachment of electrophilic/nucleophilic glass surfaces with nucleophilic/electrophilic oligonucleotides. In this method, individual oligonucleotides may be synthesized separately, purified and then they can be immobilized at defined sites on a solid surface. A number of alternative methods have been reported for the post-synthesis immobilization of oligonucleotides on a variety of surfaces (Table 1) (Ind. J. Biochem. Biophys., 40 (2003) 377; Curr. Med. Chem., 8 (2001) 1213; Curr. Pharm. Biotechnol., 4 (2003) 379). Recently, Kumar and Gupta (Bioconjugate Chemistry, 14 (2003) 507) developed a simple method to construct oligonucleotide array on polymer surfaces, using commonly available reagents and chemistry with good efficiency and accuracy. The method involves the generation of hydroxyl functionalities, followed by their activation with tresyl chloride. The activated surface in the subsequent reaction is used to covalently immobilize oligonucleotides having mercaptohexyl- or aminohexyl functionalities to create oligonucleotide array. The constructed oligonucleotide arrays were successfully used to analyze oligonucleotides by hybridization technique.
TABLE 1Immobilization of oligonucleotidesFunctional group onModification onS. No.Supportsupportoligonucleotides1.GlassThiol5′-Disulfide2.GlassIsothiocyanateAmine3.GlassAldehydeAmine4.GlassMercaptoalkylMaleimide5.GlassBromoacetamidePhosphorothioate6.Glassp-AminophenylCarboxyl7.Glass/SiliconEpoxideAmino8.Glass/polypropyleneN-Hydroxysuccinimidyl- or5′ or 3′-Aminoimidoesters9.Silanized glassAmino5′-Thiol10.Polyacrylamide onAmino or aldehyde3′-Amino or aldehydeglass11.Glass coated withCyanuric chloride3′- or 5′-Alkylaminopolyethyleneimine12.PlasticAcrylic groups5′-Acrylamide13.CPG, PS, Sephacryl,Carboxylic, alkylamino on5′-Aminoalkyl or 5′-chondroitinCPGphosphorylated14.SiliconMaleimideThiol15.Quartz/GoldMaleimideThiol16.GoldDisulfideThiolIn yet another approach, Strother et al. (Nucleic Acids Res., 2000, 28, 3535-41) described a method for attaching the oligonucleotides to silicon surfaces, which were functionalized with t-Boc protected 10-aminodec-1-ene under the influence of UV light. After attachment, t-Boc group was removed and the resulting amino groups were coupled to thiolated oligonucleotides, using a heterobifunctional crosslinker, SSMCC (sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) to generate oligonucleotide arrays. The density of immobilized oligonucleotides was controlled by varying the attachment of t-Boc-10-aminodec-1-ene and partially allowing the deprotection of t-Boc group from t-Boc-10-aminodec-1-ene. It has been observed in studies related to construction of oligonucleotide arrays that glass surfaces coated with strongly electrophilic surfaces possess poor shelf life; hence, surfaces with groups such as aminoalkyl and mercaptoalkyl have mostly been used for this purpose. In a recent method, the attachment of an oligonucleotide modified with phosphorothioates in its backbone to a glass surface with bromoacetyl functionalities has been demonstrated. The oligonucleotide reported here contains a hairpin stem-loop structure, which serves as lateral spacers between neighbouring oligodeoxynucleotides and as a linker arm between the glass surface and the single stranded sequence of interest. The main advantage of this method is that both 3′- and 5′-ends are not modified, thus making them favorable for various enzymatic and labeling reactions.
In a slightly different approach, a protocol using photochemical immobilization technique has been developed. The method involves the covalent linking of the oligonucleotides to the surface during irradiation. Traditionally, psoralens, benzophenone, azides and carbenes are used for photochemical immobilization reactions, however, as these photoprobes suffer from several inherent drawbacks, anthraquinone has been employed, as in its excited state it can react with almost any C—H containing substrate. Using this conjugated system, oligonucleotides can be immobilized on surfaces such as polystyrene, polycarbonate, polypropylene, Teflon and silylated glass, etc. As discussed above, a variety of surface chemistries have been developed for making synthetic oligonucleotide microarrays on solid- or polymeric surfaces. The production and optimal performance of these arrays depends on some factors. One of them is a linker required to create a suitable distance between surface and the oligonucleotide sequence that is to be used for hybridization experiments; the distance minimizes the steric hindrances with the incoming molecules as well as provides accessibility to them. In some cases, polyethylene glycol and oligothymidines have been employed as spacers. Other factors include physical and chemical properties of surface, derivatization of slides with suitable functional groups, incorporation of suitable modified functional groups on oligonucleotides, density of oligonucleotides on the surface, delivery of tiny volumes of spotting solution, the blocking of unreacted functional groups on the surface, length and type of target DNA molecules, hybridization and washing conditions, etc. Another problem related to uniform distribution of spotted oligonucleotide has been addressed by mixing a suitable solvent with properties, such as good wettability and low evaporation rate; betaine and dimethylsulfoxide are the most commonly used reagents for this purpose.
Most of these methods utilize modified oligonucleotides either at 3′- or 5′-end to prepare oligonucleotides arrays on the polymeric surfaces. Mostly, aminoalkyl, mercaptoalkyl, carboxyl, aldehyde, phosphate groups in oligomers are required, where one has to require either phosphoramidite reagents or engineered polymer supports to generate desired functionalities. Finally, these modified oligomers are immobilized on a variety of polymer surfaces with appropriate reactive functionality in the presence or absence of a suitable coupling reagent.